A bioassay to measure energy metabolism in mouse colonic crypts, organoids, and sorted stem cells

Abstract

Evidence suggests that targeting cancer cell energy metabolism might be an effective therapeutic approach for selective ablation of malignancies. Using a Seahorse Extracellular Flux Analyzer, we have demonstrated that select environmental agents can alter colonic mitochondrial function by increasing respiration-induced proton leak, thereby inducing apoptosis, a marker of colon cancer risk. To further probe bioenergetics in primary intestinal cells, we developed methodology that can be modified and adapted to measure the bioenergetic profiles of colonic crypts, the basic functional unit of the colon, and colonic organoids, an ex vivo 3D culture of colonic crypts. Furthermore, in combination with the MoFlo Astrios High-Speed Cell Sorter, we were able to measure the bioenergetic profiles of colonic adult stem and daughter cells from Lgr5-EGFP-IRES-creER^T2 transgenic mice. We examined the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a full arylhydrocarbon receptor agonist, known to affect gastrointestinal function and cancer risk, on the bioenergetic profiles of intestinal epithelial cells. Mouse colonic crypts, organoids, or sorted single cells were seeded onto Matrigel-precoated Seahorse XF24 microplates for extracellular flux analysis. Temporal analyses revealed distinct energy metabolic profiles in crypts and organoids challenged with TCDD. Furthermore, sorted Lgr5⁺ stem cells exhibited a Warburg-like metabolic profile. This is noteworthy because perturbations in stem cell dynamics are generally believed to represent the earliest step toward colon tumorigenesis. We propose that our innovative methodology may facilitate future in vivo/ex vivo metabolic studies using environmental agents affecting colonocyte energy metabolism.

the colonic epithelial lining represents one of the most intensively self-replenishing organs in mammals. Cell homeostasis is sustained by crypt-resident multipotent stem cells (1). Using a recently described three-dimensional culture system (4, 5), we were able to monitor the growth of mouse colonic crypts, stem cell self-renewal, and differentiation in vitro (9). This is significant because adult colonic stem cells are the cells of origin for colon tumors (4). Cancer cells are typically subject to profound metabolic alterations, including the “Warburg effect” (24) wherein most cancer cells predominantly produce energy by a high rate of aerobic glycolysis. Recent studies suggest that targeting cancer cell energy metabolism might be a new and very effective therapeutic approach for selective ablation of malignancies (12, 19, 20).

Mitochondrial function has traditionally been assessed by using Clark-type electrode probes for measuring oxygen consumption, luminescent ATP assays for quantification of total energy metabolism, and MTT or alamarBlue for determination of metabolic activity. These techniques, however, are labor intensive, cumbersome, and/or relatively inaccessible for many laboratories. Recent development of the Seahorse XF Extracellular Flux analyzer has afforded investigators the ability to measure intact cell bioenergetic profiles (BEPs) in real time (25). This relatively new streamlined, label-free assay system simultaneously measures the two major energy-producing pathways of the cell, mitochondrial respiration (oxygen consumption) and glycolysis (extracellular acidification), in a highly sensitive microplate format. Cellular oxygen consumption (respiration) and proton excretion (glycolysis) induce rapid, easily measurable changes to the concentrations of dissolved oxygen and free protons that are assessed every few seconds by solid-state sensor probes residing 200 μm above the cell monolayer. Following the addition of various mitochondrial inhibitors, several mitochondrial functional parameters can be determined and the entire cell BEP can be quantified (Fig. 1). However, most of the applications to date have been carried out with use of cell lines or isolated mitochondria because of the need to adhere cells to the bottom of multichambered microplates. This step is typically followed by repeated mixing procedures throughout the course of the analysis (∼2–3 h). Although an Islet Capture Microplate with a 125-μm pore size capture screen has been recently developed, it is not suitable for most floating cells and small size tissue samples. To date, there is a dearth of studies utilizing primary cultures, especially intestinal cells/tissues, probably due to the fragility of these cultures and the unique growth condition required to sustain specific cell populations. To overcome these obstacles, we have developed a Matrigel-based methodology for plating freshly isolated colonic crypts, cultured colonic organoids, and adult colonic stem cells to accommodate the extracellular flux measurements. Furthermore, by adjusting growth factor levels in the isolation/incubation medium, we are able to maintain the viability of stem cells ex vivo, during the measurement of bioenergetic profiles.

Fig. 1.

Representative mitochondrial function parameters generated by using the Seahorse XF24 Analyzer. Mouse colonic samples (crypts, organoids, or cells) were sequentially challenged with an ATP synthetase inhibitor (oligomycin), respiratory chain uncoupler [carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP)], and complex I inhibitor (rotenone). The profile was recorded as oxygen consumption rate (OCR) throughout the analysis. Based on the different OCR responses to specific electronic transfer chain inhibitors, basal respiration capacity, ATP turnover, proton leak, maximum respiratory capacity, and reactive oxygen species were calculated as indicated.

The arylhydrocarbon receptor (AhR) is highly expressed in multiple organs and tissues, and there is increasing evidence that the AhR plays an important role in cellular homeostasis and disease (21). The most high-affinity AhR agonist is the notorious environmental and industrial toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD induces a characteristic semiacute wasting syndrome in several animal models and represents a puzzling and dramatic perturbation of the regulatory systems for energy balance (17). TCDD also modulates the phenotype of various cancer cell types, including but not limited to breast, ovarian, liver, and colon cancer cells (13, 15, 21, 26). To further assess the effects of TCDD with respect to energy balance, we optimized methodologies to measure the real-time BEP of mouse colonic crypts and colonic organoid cultures exposed to TCDD. In addition, using high-speed cell sorting, we measured for the first time the distinct oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), representing oxidative phosphorylation/glycolysis profiles, in adult colonic stem cells and daughter cells freshly isolated from Lgr5-EGFP-IRES-creER^T2 transgenic mice.

MATERIALS AND METHODS

Animals

C57BL/6 mice were used for TCDD studies. Lgr5-EGFP-IRES-creER^T2 transgenic mice, originally generated by H. Clevers at the Hubrecht Institute University Medical Center, Utrecht, The Netherlands (2), were used for stem cell studies. All procedures adhered to the guidelines approved by Public Health Service and the Institutional Animal Care and Use Committee at Texas A&M University.

TCDD Treatment

For in vivo dosing studies, mice were orally gavaged with TCDD (25 μg/kg body wt) or corn oil (vehicle control) daily for 4 consecutive days. Colonic crypts were subsequently isolated and bioenergetic profiles were measured by using the Seahorse bioanalyzer as described in detailed protocols. For ex vivo analyses, mouse colonic crypts were isolated and cultured to mature organoids in complete medium containing Advanced DMEM/F12, GlutaMax (2 mM), 100 U/ml penicillin, 100 μg/ml streptomycin, recombinant mouse EGF (50 ng/ml), N2 supplement (1×), and B27 supplement (1×) (all from LifeTechnologies, Grand Island, NY); LDN-193189 (0.2 μM) (Cellagen Technology, San Diego, CA); R-Spondin (500 ng/ml) (Sino Biological, Beijing, China); N-acetylcysteine (1 μM) and HEPES (10 mM) (Sigma, St. Louis, MO); and Wnt conditioned medium (1:1 dilution) (9, 22) for 5 days. TCDD (1 nM) or DMSO (vehicle control) were added to the cultures for 3 additional days. Organoids were then harvested, and bioenergetic profiles were measured.

Stem Cell Analyses

Colonocytes were isolated from Lgr5-EGFP-IRES-creER^T2 transgenic mouse colons followed by high-speed cell sorting on a Beckman Coulter MoFlo Astrios to selectively collect green fluorescent protein (GFP)-high (stem cell) and GFP-low (daughter cell) populations as previously described (8). Gating strategy details for sorting Lgr5-GFP-high and low cells are described in Fig. 2. For gating purposes, colonocytes isolated from littermate wild-type mice were used to exclude GFP-negative cells. Bioenergetic profiles of stem cells vs. daughter cells were subsequently measured.

Fig. 2.

Gating parameters for fluorescence-activated cell sorting. Gates are set to exclude cellular debris (A), exclude propidium iodide (PI)-positive cells (B), exclude cell doublets (C) and exclude nonepithelial cells (D). E: gates are set to collect green fluorescent protein (GFP)-high (stem cells) and GFP low-expressing cells (daughter cells). F: colons from wild-type mice display no GFP-expressing cells. SSC, side scatter; FSC, forward scatter; EPCAM, epithelial cell marker.

Statistical Analysis

Data were analyzed by t-tests with significance at P < 0.05. All data are presented as means ± SE, and all analyses were conducted by use of the Prism 6 program (GraphPad Software, La Jolla, CA).

DETAILED PROTOCOLS

Colonic Crypt Isolation

Processing time ∼60 min.

1. Prepare ADF⁺ medium: Advanced DMEM/F12 medium (LifeTechnologies, no. 12634-010) supplemented with 1% Glutamax (LifeTechnologies, no. 35050-061), 1% penicillin-streptomycin (LifeTechnologies, no. 15140-148), and 1% HEPES (Sigma, no. H0887). Can be stored at 4°C up to 2 wk.

2. Make fresh 20 mM EDTA in Ca²⁺/Mg²⁺-free HBSS (Mediatech no. 21-021-CV), adjust to pH 7.4. Warm to 37°C in a water bath.

3. Set up an eversion station by positioning a disposable 10-ml syringe upright on a rack and attach a disposable gavage needle (Soloman Scientific no. FTP-20-38) onto the syringe.

4. Fill a 5-ml syringe with cold HBSS and attach a gavage needle (Popper & Sons, no. 7922).

5. For each tissue, prepare 3 × 50-ml conical tubes containing cold HBSS (∼30 ml/tube, with 1 tube also containing 0.5% penicillin-streptomycin).

6. Euthanize the mouse with CO₂ followed by cervical dislocation.

7. Remove the colon and place in a cup of cold HBSS.

8. Rinse the tissue by swishing in cold HBSS and remove excess fat using a forceps.

9. Use the preloaded 5-ml syringe (from step 4) to perfuse the colon to flush out feces.

10. Gently thread the proximal end of the colon (wider) onto the disposal gavage needle. Once the entire colon passes through the tip of the needle, tie the distal end (narrower) onto the needle with a piece of string and cut off the extra length of string. Evert the tissue by grasping the lower part of the colon with two forceps and gently pulling it upward until it is completely everted. Place the tissue attached to the gavage needle into the conical tube containing 30 ml cold HBSS with 0.5% penicillin-streptomycin. Keep on ice.

11. Vortex the colon (in the conical tube with cold HBSS) at maximum speed, 6 × 5 s each, to remove remaining debris, making sure the tissue is untangled between/after vortexing cycles.

12. Use a forceps to transfer the colon to another conical tube containing 30 ml cold HBSS. Vortex at maximum speed 3 × 5 s each.

13. Transfer colons to the prewarmed 20 mM EDTA/HBSS in a 50-ml conical tube. Incubate at 37°C in a water bath for 30 min.

14. Following incubation, transfer the tissue to a conical tube containing ∼30 ml cold HBSS and vortex at maximum speed 8 × 5 s each to release crypts. Take 10-μl aliquots and apply to a petri dish; check under an inverted microscope to see the yield of crypts dislodged from the tissue. Continue the isolation process using additional vortexing if necessary.

15. Remove residual colon tissue on needle and discard. Add 3 ml FBS to the tube containing crypts to yield a final 10% FBS/HBSS solution and spin down the crypts at 125 g for 3 min.

16. Aspirate solution and resuspend crypts with ∼13 ml cold ADF⁺ and transfer to a 15-ml conical tube.

17. Centrifuge at 70 g for 2 min.

18. Repeat the ADF⁺ wash 2–3× to help remove single cells, pipetting up and down multiple times.

19. Take an aliquot and count. Typical yield ∼80–120,000 crypts from 1 mouse colon.

20. The isolated crypts can be used for organoid culture, single cell isolation, or directly used for Seahorse Extracellular Flux XF24 bioanalyzer measurements.

21. For BEP analysis, resuspend the crypts at a density of 250 crypts/50 μl Seahorse-ADF medium (Seahorse XF Assay Medium, Seahorse Bioscience, no. 100965-000) supplemented with 17.5 mM glucose (Sigma, no. G8769), 2 mM Glutamax (LifeTechnologies, no. 35050), 1 mM sodium pyruvate (Sigma, no. S8636), and 1% penicillin-streptomycin (LifeTechnologies, no. 15140-148) adjusted to pH to 7.4.

Organoid Culture

Processing time ∼20 min.

1. Prechill 200 and 1,000 μl pipet tips at 4°C.

2. Thaw the growth factor reduced basement membrane matrix Matrigel (Corning, no. 356231) on ice, and warm up 24-well culture plates (Costar, no. 3524) in a 37°C cell culture incubator at least 30 min prior to finishing the crypt isolation.

3. Prepare complete organoid medium by adding the following growth factors to ADF⁺ medium: EGF (50 ng/ml) (LifeTechnologies, no. PMG8043), LDN-193189 (0.2 μM) (Cellagen Technology, no. C5361-2s), R-Spondin (500 ng/ml) (Sino Biological), N2 supplement (1×) (LifeTechnologies, no. 17502-048), B27 supplement (1×) (Life Technologies, no. 12587-010), N-acetylcysteine (1 μM) (Sigma, no. A7250) and Wnt conditioned medium (1:1 dilution) as previously described by Barker et al. (3).

4. Aliquot the crypts from step 20 in the Colonic Crypt Isolation protocol (∼500-1,000 crypts per well) to a 15-ml conical tube, fill the tube with ADF⁺ to ∼10 ml to resuspend the crypts well, and spin down at 100 g for 3 min.

5. Thoroughly discard the supernatant and keep the crypts cold at all times. Place the prechilled pipet tip boxes on ice and use the chilled pipet tips to gently resuspend the pellet to a density of 500-1,000 crypts/50 μl Matrigel per well. Avoid bubbles.

6. Seed 50 μl of Matrigel/crypt mix to the center of each well of prewarmed 24-well plate, and incubate for 5–10 min in 37°C incubator until solidified. The droplet of Matrigel should remain in the center of the well and not spread.

7. During the Matrigel solidification step, prewarm the complete organoid medium to 37°C.

8. Once the Matrigel is solidified, add 500 μl warm complete organoid medium to each well without touching the Matrigel mound. The medium should barely cover the Matrigel/crypt mound.

9. Incubate at 37°C in a CO₂ incubator. The plate is now ready for application of the ex vivo treatments. Change complete medium every 2–3 days as needed.

In general, the yield of viable crypts is ∼10%. Live crypts will start budding after 2–3 days in culture.

Organoid Harvest

Processing time ∼15 min.

10. Place organoid culture plates on ice. Carefully aspirate and discard culture medium, add 0.5 ml ice-cold ADF⁺, and mechanically break up the Matrigel by pipetting up and down multiple times using a 1,000-μl pipet. Transfer the dissociated Matrigel into a 15-ml conical tube.

11. Wash the well with ice-cold ADF⁺ (0.5 ml) to recover most of the organoids and add to the 15-ml tube. Organoids tend to grow more at the edge of Matrigel. Check the culture wells under microscope to ensure that all organoids have been harvested.

12. Fill the 15-ml conical tube with cold ADF⁺ to at least 10 ml to dissociate and wash the organoids from the Matrigel.

13. Centrifuge at 200 g for 4 min.

14. Remove supernatant and resuspend in 1 ml Seahorse-ADF medium.

15. Using a 1-ml syringe, pass the suspension through a 20-gauge needle 5× on ice to break up the organoids into small pieces.

16. Take 5 μl and apply to a small petri dish to count the number of organoid pieces.

17. Resuspend samples to a density of 250 organoids/50 μl Seahorse-ADF medium/well. The sample is now ready for BEP analysis.

Single Cell Isolation and Fluorescence-Activated Cell Sorting

Processing time ∼45 min for single cell isolation, ∼1 h for cell sorting.

1. Precoat 5-ml polypropylene tubes (BD, no. 352063) with 5 ml of 2% FBS/ADF⁺ the day before cell sorting.

2. Prewarm 0.25% Trypsin-EDTA (LifeTechnologies, no. 25200-056) to room temperature.

3. Make fresh 500 mM butyrate working stock by mixing 0.055 g butyrate (Sigma, no. B5887) in 1 ml PBS, filter sterilize through 0.2 μm Acrodisc Syringe Filter (PALL Life Sciences, no. PN4454).

4. Prepare single-cell cocktail: ADF⁺ medium containing 10 μM Y-27632 (Sigma, no. Y0503), 1 μM N-acetylcysteine, 0.5% BSA, 200 U/ml DNase (Sigma no. D5025), 2 mM EDTA and 5 mM butyrate.

5. Resuspend crypts from step 20 in the Colonic Crypt Isolation protocol in 5 ml of 0.25% trypsin-EDTA; incubate at 37°C in a CO₂ incubator for 7 min.

6. Pass suspension through a 20-gauge needle 3× to aid dissociation. Let sit 1 min at room temperature. Pass the sample through the needle one last time to break up cell clusters.

7. Add 10 ml ice-cold 5% FBS/ADF⁺ to stop the trypsin digestion reaction. Pass contents through a 20-μm Partec filter (CellTrics, no. 04-0042-2315). Multiple filters may be needed since small clumps of cells or crypts can clog the filter.

8. Transfer the filtered cell suspension to a 50-ml conical tube. Add an additional 10 ml ADF⁺, 5 μl of 10 mM Y-27632, 20 μl of 500 mM butyrate to the cell suspension. The butyrate provides an energy source for the colonocytes.

9. Take an aliquot to count the number of cells.

10. Centrifuge the cell suspension at 500 g for 3 min 4°C. A very small pellet should be visible. If not, spin again at 550–600 g for 1 min.

11. Carefully aspirate the supernatant and resuspend the cell pellet to a density of 2–4×10⁶ cells/ml, depending on the requirements of the flow cytometer, in single-cell cocktail. Keep samples on ice.

12. Just before starting cell sorting, filter cell suspensions again using a 20-μm Partec filter into the precoated 5-ml tube. Rinse the filter with a small volume (∼100 μl) of single-cell cocktail to recover most of the cells.

13. Add propidium iodine solution (5 μl/500 μl cell suspension) (Miltenyi Biotec, no. 130-093-233) to the cell suspension to allow exclusion of dead cells from cell sorting.

14. Sort the GFP high, low, and negative cell populations using a MoFlo Astrios High-Speed Cell Sorter (Beckman Coulter, no. A66831).

15. Collect sorted cells into 2× growth factor enriched Seahorse-ADF medium (GF-Seahorse-ADF): Seahorse-ADF medium containing EGF (100 ng/ml), LDN-193189 (0.4 μM), R-Spondin (1 μg/ml), N2 supplement (2×), B27 supplement (2×), Y-27632 (20 μM), and fresh butyrate (10 mM). Sorted cells, collected mostly in PBS, will be diluted in the 2× medium to final 1×. Keep sorted cells on ice.

16. Transfer sorted cells to a 1.7-ml epi-tube. Count cell number and viability. Centrifuge at 500 g for 3 min at 4°C. Resuspend cell pellets to a density of 40,000 cells/50 μl 1× GF-Seahorse-ADF medium/well. 1× GF-Seahorse-ADF medium: Seahorse-ADF medium containing EGF (50 ng/ml), LDN-193189 (0.2 μM), R-Spondin (500 ng/ml), N2 supplement (1×), B27 supplement (1×), Y-27632 (10 μM), and fresh butyrate (5 mM). Cells are now ready for BEP analysis.

Seahorse XF24 Extracellular Flux BEP Measurement

The day before BEP analysis.

Preparation of Matrigel-precoated plates. Processing time ∼30 min excluding the 1 h incubation.

1. Prepare Seahorse-ADF medium.

2. Dilute Matrigel 1:10 (vol/vol) in Seahorse-ADF medium. Add 50 μl/well to the Seahorse XF24 cell culture microplate.

3. Incubate at room temperature for at least 1 h.

4. Seal with Parafilm. Store at 4°C overnight. Can be stored for up to 1 wk.

5. Prewarm Seahorse XF24 Extracellular Flux Analyzer (Seahorse Bioscience, Billerica, MA) to 37°C. At least 3 h before analyzing samples, overnight stabilization preferred.

6. Hydrate the XF 24 cartridge plate by adding ∼1 ml/well of XF Calibrant (Seahorse Bioscience, no. 100840-000). Incubate at 37°C in Seahorse XF Plate Prep Station or a non-CO₂ incubator. Can be prepared 3 days ahead; make sure the cartridges are well submerged in the XF Calibrant. If the cartridges dry out, the measurement will not be accurate.

The day of analysis.

Processing time ∼45 min hands-on time, and ∼130 min machine run time.

Set up the assay plate template and program according to the Seahorse XF24 Bioanalyzer “Assay Wizard” section. The programs used are shown in the Program protocol for crypts/organoids and Program protocol for isolated cells sections below.

1. Prepare 10× mitochondrial inhibitor compounds by diluting the stock solutions in Seahorse-ADF medium (for crypts and organoids) or 1× GF-Seahorse-ADF medium (for single cells), and pH to 7.4.

The 10× working compounds for crypts, organoids, and cells are listed as follows.

Crypts: oligomycin (20 μM) (Sigma, no. O4867), FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone; 5 μM) (Sigma, no. C2920), and rotenone (50 μM) (Sigma, no. R8875).

Organoids: oligomycin (20 μM), FCCP (25 μM), and rotenone (50 μM).

Isolated cells: oligomycin (20 μM), FCCP (125 μM), and rotenone (100 μM).

2. Take the hydrated cartridge plate out of the incubator. Add 50 μl oligomycin to port A, 55 μl FCCP to port B, and 60 μl rotenone to port C of each treatment well. For basal and unused wells, add medium instead of the mitochondrial inhibitors.

3. Incubate at 37°C in the Seahorse XF Plate Prep Station or a non-CO₂ incubator ∼30–60 min before the assay.

4. Warm up the diluted Matrigel-precoated cell culture microplate and Seahorse-ADF medium (for crypts and organoids) or 1× GF-Seahorse-ADF medium (for single cells) to room temperature ∼30–60 min before seeding crypts, organoids, or cells. Avoid warming up the Matrigel-coated plate too early, because the coating may dry out.

5. Adjust samples to an appropriate seeding density: crypts (250 crypts/50 μl) or organoids (250 organoids/50 μl) in ADF-Seahorse medium; single colonocytes (minimum 40,000 cells/50 μl) in 1× GF-Seahorse-ADF medium.

6. Aspirate the diluted Matrigel from the cell culture microplate wells. Wash with warm Seahorse-ADF medium once (∼200 μl/well). Add 50 μl of Seahorse-ADF (or 1× GF-Seahorse-ADF medium) to each well, then gradually add 50 μl of each sample to the well in a circular motion to evenly distribute the samples in the well. The preaddition of 50-μl medium acts as a coating/diluting buffer to help the samples disperse more evenly. The even distribution of cells in the well will provide a more accurate measurement.

7. Immediately incubate the samples at 37°C in the Seahorse XF Plate Prep Station or a non-CO₂ incubator for 30 min.

8. Start the program by first calibrating the cartridge plate during the 30-min incubation. Transfer the cartridge plate from the Seahorse XF Plate Prep Station or non-CO₂ incubator, and place inside the Analyzer for calibration. Calibration takes ∼30 min.

9. Meanwhile, keep the Seahorse-ADF (or 1× GF-Seahorse-ADF) medium at 37°C in a water bath to maintain the same temperature as the incubated samples.

10. After 30 min, take the samples out of incubator. Add an additional 400 μl 37°C medium to each well by gently adding from the top corner of the well, to avoid agitating the cells which are loosely attached to the wells.

11. Return the plate to the Seahorse XF Plate Prep Station or 37°C non-CO₂ incubator for ∼10 min to equilibrate the cells/medium. Then transfer the cell plate into the XF24 Analyzer and continue the program.

At the end of the assay, if you want to save the cell culture microplate for normalization, perform the following steps:

1. Remove the sample microplate from the XF24 soon after the assay is finished.

2. Gently remove the medium by inverting and tapping the microplate onto a paper towel.

3. Apply the cover to the microplate. Tape all four sides to ensure the lid is attached to the plate for cell normalization.

4. Store at −80°C.

5. Within 1 mo, follow the protocol for CyQuant Cell Proliferation Assay Kit (LifeTechnologies, no. C7026) to measure the cell density for normalization.

Seahorse XF24 Extracellular Flux Analyzer Programs for Crypts/Organoids and Single Cells

Program protocol for crypts/organoids.

Calibrate

Equilibrate

Loop start: 3×

Mix: 3 min

Wait: 2 min

Measure: 3 min

Loop end

Inject: port A (oligomycin)

Loop start: 3×

Mix: 3 min

Wait: 2 min

Measure: 3 min

Loop end

Inject: port B (FCCP)

Loop start: 3×

Mix: 3 min

Wait: 2 min

Measure: 3 min

Loop end

Inject: port C (rotenone)

Loop start: 3×

Mix: 3 min

Wait: 2 min

Measure: 3 min

Loop end

End

Program protocol for isolated cells.

Calibrate

Equilibrate

Loop start: 3×

Mix: 2 min

Wait: 2 min

Measure: 5 min

Loop end

Inject: port A (oligomycin)

Loop start: 3×

Mix: 2 min

Wait: 2 min

Measure: 5 min

Loop end

Inject: port B (FCCP)

Loop start: 3×

Mix: 2 min

Wait: 2 min

Measure: 5 min

Loop end

Inject: port C (rotenone)

Loop start: 3×

Measure: 2 min

Wait: 2 min

Measure: 5 min

Loop end

End

RESULTS

Assessment of Colonocyte Viability and Optimization of Extracellular Flux Analysis

A representative Seahorse XF24 bioenergetic profile is shown in Fig. 1. Representative microscopy images of mouse colonic crypts before and after Seahorse Extracellular Flux Analyzer measurement are shown in Fig. 3. Although single cells gradually dislodged from the intact crypts as the assay time increased (Fig. 3, A–C), the OCR was fairly stable over time (Fig. 3D). This indicates that despite the morphology change, basal energy metabolism was quite stable, indicating acceptable cell viability. In addition, parallel samples were cultured in a non-CO₂ incubator to mimic the analyzer conditions and cell viability was measured by using the Live/Dead Cell Viability Assay (LifeTechnologies, no. L3224). Typically, the viability was 90% at the end of the bioassay (∼3.5 h from the initial seeding of crypts). Similar results were observed in the organoid cultures, where comparatively fewer single cells dislodged from the organoids (Fig. 3G). This may be due to the fact that organoids had already been cultured for several days and were therefore more resistant to the additional manipulation associated with the Seahorse analyzer. Despite these modest phenotypic differences, both crypts and organoids exhibited a stable basal OCR, indicating that the isolation and culturing protocols did not negatively impact cellular basal energy metabolism. Similarly, both cell morphology and real-time bioenergetic profiles of sorted single cells indicated a high level of viability (Fig. 3K). In subsequent experiments, bioenergetic profiles were generated following treatment with mitochondrial inhibitors (oligomycin, ATP synthetase inhibitor; FCCP, uncoupler; rotenone, Complex I inhibitor) in the three distinct colonic isolates, i.e., crypts, organoids, and sorted stem cells (Fig. 3, I–N). The comparable profiles suggest that our method is suitable for measuring energy metabolism, e.g., OCR and ECAR, in the various colonocyte samples.

Fig. 3.

Cell viability and bioenergetic profiles of colonic crypts, primary organoid cultures, and isolated stem cells. Representative images of freshly isolated crypts at time 0 (before plating) (A), 30 min after seeding in diluted Matrigel-precoated cell culture microplates (B), and 3.5 h after completion of the assay (C). D: representative basal OCR of crypts throughout the entire extracellular flux analysis. Representative images of 5-day-old organoids used for basal energy metabolism measurements at time 0 (E), 30 min (F), and 3.5 h (G). H: representative basal OCR of organoids throughout the entire extracellular flux analysis. Representative bioenergetic profiles [OCR, I–K; extracellular acidification rate (ECAR), L–N] of colonic crypts, organoids, and adult stem cells under basal conditions and following treatment with mitochondrial inhibitors (oligomycin, FCCP, rotenone).

TCDD Treatment Alters Colonocyte Bioenergetic Profiles

To determine the in vivo effects of TCDD on the intestinal tract, C57BL/6 mice were gavaged with TCDD (25 μg/kg body wt) or control (corn oil) for 4 consecutive days. Colonic crypts were then isolated and the BEP was measured (Table 1). For comparative purposes, an ex vivo TCDD study was conducted, in which mouse crypts were plated, grown for 5 days to establish a mature organoid culture in vitro and subsequently incubated with TCDD (1 nM) or control (DMSO-vehicle) for an 3 additional days. Subsequently, organoids were harvested and the BEP was measured (Table 2). Despite the different regimens, i.e., in vivo and in vitro treatment, TCDD had a similar effect on the various mitochondrial parameters, including basal OCR-to-ECAR ratio (OCR/ECAR), ATP turnover, proton leak, and reactive oxygen species. These findings demonstrate the organoid model can recapitulate the in vivo effects of TCDD.

Table 1.

Bioenergetic profiles of mouse colonic crypts following TCDD exposure

	Control	TCDD	P Value
Basal OCR, pmol/min	132.0 ± 23.2	198.9 ± 19.8	0.043
Basal ECAR, mpH/min	8.0 ± 0.8	7.6 ± 1.0	0.779
OCR/ECAR	19.7 ± 2.5	27.8 ± 2.5	0.035
Respiration (pmol O2/min) utilized for
ATP turnover	52.5 ± 9.9	97.2 ± 15.0	0.027
Proton leak	112.0 ± 14.5	73.7 ± 10.0	0.038
Maximal respiration capacity	172.8 ± 25.4	204.1 ± 33.3	0.460
Reserved respiration capacity	31.7 ± 9.1	78.1 ± 24.2	0.113
ROS	16.8 ± 2.9	29.6 ± 3.2	0.008

Values are means ± SE (n = 8–12). C57BL/6 mice were gavaged with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at 25 μg/kg body weight or vehicle control (corn oil) daily for 4 consecutive days. OCR, oxygen consumption rate; ECAR, extracellular acidification rate; ROS, reactive oxygen species. Colonic crypts were subsequently isolated and bioenergetic profiles were measured by using the Seahorse bioanalyzer as described in materials and methods.

Table 2.

Effect of TCDD on mouse colonic organoid mitochondrial bioenergetic profiles

	Control	TCDD	P Value
Basal OCR, pmol/min	314.8 ± 89.1	201.2 ± 58.0	0.163
Basal ECAR, mpH/min	14.8 ± 5.9	4.6 ± 1.5	0.088
OCR/ECAR	5.6 ± 3.7	20.1 ± 6.2	0.037
% Basal OCR utilized for
ATP turnover	43.0 ± 5.9	67.1 ± 5.8	0.012
Proton leak	57.0 ± 5.9	38.4 ± 7.1	0.039
Maximal respiration capacity	172.4 ± 7.5	137.1 ± 27.8	0.107
Reserved respiration capacity	72.4 ± 7.5	23.3 ± 16.6	0.008
ROS	42.9 ± 5.5	63.8 ± 13.9	0.086

Values are means ± SE (n = 4–6). Mouse colonic crypts isolated from chow-fed C57BL6 mice were cultured to mature organoids for 5 days. Colonic organoid cultures were then incubated with TCDD (1 nM) or control (DMSO-vehicle) for an additional 3 days. Organoids were harvested and bioenergetic profiles were measured.

Contrasting Bioenergetic Profiles of Colonic Adult Stem Cells Compared with Differentiated Daughter Cells

Colonic intestinal stem cells and differentiated daughter cells from Lgr5-EGFP-IRES-creER^T2 mice were isolated and their respective bioenergetic profiles were analyzed. As show in Table 3, stem cells and daughter cells had significantly (P < 0.05) different basal OCR, proton leak, maximal respiration capacity, and OCR/ECAR ratio. Importantly, the low OCR/ECAR ratio observed in stem cells indicates a decreased oxidative phosphorylation phenotype, consistent with a Warburg metabolic profile, i.e., decreased mitochondrial metabolism and increased glycolytic flux. The comparatively low basal OCR phenotype in Lgr5⁺ colonic stem cells vs. daughter cells is consistent with a recent report documenting energy metabolism in human pluripotent stem cells, which exhibited a lower OCR relative to their differentiated cell counterparts (23). In addition, the lower proton leak in stem cells suggests a reduced apoptotic activity since mitochondrial proton leak is generally positively correlated with programmed cell death (10). This phenotype is also consistent with reports that stem cells are resistant to apoptosis (7).

Table 3.

Comparison of mitochondrial bioenergetic profiles in adult mouse colonic stem cells and daughter cells

	Stem Cells	Daughter Cells	P Value
Basal OCR, pmol/min	164.5 ± 15.7	248.5 ± 20.9	0.004
Basal ECAR, mpH/min	5.8 ± 1.1	6.2 ± 0.8	0.754
OCR/ECAR	33.9 ± 5.0	59.6 ± 12.2	0.042
Respiration (pmol O2/min) utilized for
ATP turnover	23.6 ± 8.9	16.7 ± 3.9	0.418
Proton leak	9.2 ± 1.6	23.2 ± 2.7	0.003
Maximal respiration capacity	18.8 ± 3.1	59.3 ± 6.6	0.001
Reserved respiration capacity	10.8 ± 6.4	23.8 ± 6.4	0.171
ROS	16.2 ± 5.4	7.9 ± 5.0	0.280

Values are means ± SE (n = 8–9). Colonic epithelial cells were isolated from Lgr5-EGFP-IRES-creER^T2 transgenic mice. GFP-positive cells were sorted to separate the GFP-high (stem cells) and GFP-low (daughter cells) populations. Bioenergetic profiles of these 2 populations are described.

TCDD Treatment Suppresses the Stem Cell Metabolic Phenotype

Figure 4 shows the basal OCR/ECAR profile of colonic crypts (Fig. 4A) and organoids (Fig. 4B) treated with TCDD. Both in vivo (crypts) and in vitro (organoids) models show the same TCDD-induced bioenergetic profiles, i.e., a significantly (P < 0.05) enhanced OCR/ECAR, indicating that the dioxin-treated cells shifted to an enhanced oxidative phosphorylation, reduced glycolytic phenotype. This type of bioenergetic profile contrasts with the aerobic glycolysis Warburg effect exhibited by cancer stem cells (24) and is therefore consistent with a suppression of stemness (Fig. 4C). This finding was corroborated by flow cytometry studies conducted in parallel, in which TCDD-treated cells exhibited a significant (48%) reduction (P < 0.05, n = 4) in the number of GFP⁺ stem cells compared with control (data not shown). This is consistent with the effect of TCDD on skin stem cell turnover (18). Collectively, these findings validate the utility of our methodology, which affords investigators the ability to measure energy metabolism in colonic crypts, organoids, and sorted stem cells.

Fig. 4.

Comparative profiles of basal OCR-to-ECAR ratios from mouse colonic crypts, cultured organoids, and sorted stem cells. Basal oxidative phosphorylation and glycolysis phenotypic profiles are shown. A: intact colonic crypts assayed immediately following isolation. B: cultured colonic crypt organoids following tetrachlorodibenzo-p-dioxin (TCDD) treatment. C: sorted adult stem cells and daughter cells from colonic crypts assayed immediately following isolation. Data represent means ± SE from n = 4–12 samples. *Significantly different, P < 0.05.

DISCUSSION

Cells constantly adjust their metabolic state in response to extracellular signals and nutrient availability to meet their demand for energy and metabolic building blocks. With the recent focus on cancer stem cell research, metabolic reprogramming is now considered a hallmark of tumorigenesis (11). Therefore, it is noteworthy that our Matrigel-based extracellular flux analysis methodology allows for the real-time monitoring of intestinal bioenergetic profiles using three-dimensional colonic crypt and organoid cultures. This novel application of organoid cultures containing epithelial and mesenchymal elements for the purpose of metabolic modeling holds great promise because it combines the accurate multilineage differentiation and physiology of in vivo systems with the facile in vitro manipulation of organoids and sorted stem cells (14). Recently, Bas and Augenlicht (6) have reported on the utilization of the Seahorse Extracellular Flux analyzer using small intestine organoids. However, their crypt isolation protocol is labor intensive, and all manipulations were carried out only using an in vitro culture system. In comparison, our crypt isolation protocol is more time efficient, i.e., less than 1 h to isolate and process multiple samples. Hence, the modified methodology allows for a broader range of applications in the field of colon cancer metabolomics. For example, researchers can perform various in vivo manipulations, including but not limited to diet, probiotics, environmental agents, toxins, and/or exercise regimens. Animals can be terminated and the in vivo effect of such manipulations on colonic crypt energy metabolism determined. These experiments can be complemented by in vitro treatments utilizing colonic organoid cultures. At the end of the treatment period, one simply harvests organoids for bioenergetic profile measurement.

Stem cells in adult tissues produce large numbers of differentiated progeny. Since transformation of adult colonic stem cells is an extremely important route toward initiating colon cancer (16), targeting stem cell energy metabolism may be a very effective therapeutic approach for cancer treatment. The Lgr5-EGFP-IRES-creER^T2 knockin mouse model allows researchers to distinguish stem cell (GFP-high) vs. daughter cell (GFP-low) populations based on GFP intensity. Combination of this transgenic mouse model with cell sorting and Matrigel-based Extracellular Flux methodology allows for the monitoring of adult stem cell energy metabolism in real time. This is a great advantage in view of the central role of stem cell metabolism in tumorigenesis. However, several factors complicate the efficiency of this “combined” methodology. For example, primary colonocytes are extremely fragile, and viability of cells gradually drops during the cell sorting procedure. Therefore, the addition of growth factors during cell isolation and cell sorting are critical in maintaining a robust OCR. In addition, the yield of GFP-high stem cells is quite variable, ranging from ∼20,000 to 80,000 stem cells per mouse colon and is affected by diet, age, and other isolation factors. A minor drawback of this methodology is that only a limited number of samples can be tested in the XF24 system at one time. However, with the recent development of the Seahorse XF96 analyzer, researchers can adjust the seeding density and test far more samples in one assay.

In summary, we propose that our innovative methodology will facilitate future in vivo/ex vivo metabolic studies using environmental agents affecting colonic crypt/stem cell energy metabolism.

GRANTS

This work was supported in part by the American Institute for Cancer Research and National Institutes of Health Grants CA129444, CA168312, and P30ES023512.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Y.-Y.F., L.A.D., S.S., and R.S.C. conception and design of research; Y.-Y.F., L.A.D., and E.S.C. performed experiments; Y.-Y.F., L.A.D., and G.A.W. analyzed data; Y.-Y.F., L.A.D., S.S., and R.S.C. interpreted results of experiments; Y.-Y.F., L.A.D., and G.A.W. prepared figures; Y.-Y.F. and L.A.D. drafted manuscript; Y.-Y.F., L.A.D., S.S., and R.S.C. edited and revised manuscript; Y.-Y.F., L.A.D., E.S.C., G.A.W., S.S., and R.S.C. approved final version of manuscript.